Perhaps the single best known medication is acetylsalicylic acid--aspirin. Aspirin has long been known to assist in the treatment of pain, swelling and fever. More recently, aspirin has been used to retard blood clotting and lower the risk of heart attacks and strokes.
While the therapeutic value of aspirin has been known for around 100 years, the therapeutic value of closely related compounds dates back far longer. See generally J. R. Cutt and D. F. Klessig, "Salicylic Acid in Plants--A Changing Perspective", Pharmaceutical Technology, May 1992, pages 26-33, the text of which is hereby incorporated by reference. For example, Hippocrates in the fourth century B.C. is believed to have prescribed the leaves of willow trees for the relief of pain during childbirth. These leaves contain salicylic acid (also referred to herein as "SA"), a naturally occurring relative of aspirin. Both can be considered part of a broader family of compounds, naturally occurring and synthetic, known as salicylates. While salicylates have long been known to exist in plants, the role that these compounds have played is only now becoming known. For many years, salicylates were classified as secondary metabolites which played no essential role in functioning of the organism. However, more recently, salicylates are gaining recognition as important factors in a number of important plant functions. An expansive, but by no means comprehensive list of plant processes which are affected by salicylates, and in particular, by the addition of salicylic acid thereto is found in Table 1.
TABLE 1 ______________________________________ Process Effect ______________________________________ Flowering + Thermogenesis + Alternative pathway + Glycolysis + Krebs cycle + Wound response - Disease resistance + Ethylene biosynthesis - Potassium ion absorption - Transpiration - Stomatal closure - Leaf abscission - Seed germination - Seed germination + Growth inhibition + Adventitious root initiation + Fruit yield + Somatic embryogenesis - Photonastic leaflet movement + Scotonastic leaflet movement - Gene regulation + ______________________________________ + = induces or enhances - = reduces or inhibits
It is interesting to note that salicylic acid has been shown to have effects on both the wound response and disease resistance of plants. In this way, plants and animals appear to share some similarities.
Although much remains to be learned about a plant's response to wounds, disease, and the attack by plant pathogens, two specific phenomena have already been observed. First, plants activate a number of a "local" responses in their attempt to restrict the spread of pathogens. This often results in the death of a very limited part of the plant immediately surrounding the site of infection. This is called the hypersensitive or local defense response.
In addition to the local defense response, many plants respond to infection by activating defenses in uninfected parts of the plant. As a result, the entire plant becomes more resistant to secondary infection. This phenomenon, sometimes termed systemic acquired resistance, can persist for some extended period of time and often confers upon the plant a resistance to unrelated types of pathogens.
It is known that adding salicylic acid to certain plants enhances their resistance to disease. It has also been shown that such additions of salicylic acid can induce the expression of the genetic material (genes) within plants to produce certain proteins related to disease resistance. Based on this information and the observation that salicylic acid is not directly toxic to most pathogens under normal conditions, it is believed that salicylic acid participates in a chain of biochemical events which ends in the production of disease combating proteins and possibly other factors or compounds. Salicylic acid may therefore be thought of as a signal molecule in the transduction pathway of plant disease resistance or a link in the chain of events leading to a plant's "immune" response. See Z. Chen and D. F. Klessig, "Identification of Soluble Salicylic Acid-Binding Protein That May Function in Signal Transduction in the Plant-Disease Response", Proc. Natl. Acad. Sci. USA 88, 8179-8183, (September 1991), the text of which is hereby incorporated by reference. The fact that when salicylic acid is added to plants, a broad number of plant functions are affected suggests that salicylates or related compounds may be effectors or signal compounds along the transduction pathway of a number of plant functions other than just disease resistance.
The present inventors have identified, purified and characterized a protein which is made by plants. This endogenous protein (endogenous meaning made by the plant) is capable of participating in the binding of salicylic acid in plants. Therefore, it is believed that the endogenous protein may be a link in the transduction pathway of various plant functions such as, for example, disease resistance. Furthermore, the inventors have cloned and sequenced a gene from tobacco which encodes this binding protein.
This discovery is important for a number of reasons. First, the identification of this binding protein, (referred to herein as either "Salicyclic Acid Binding Protein", or "SABP",), is important in gaining a further understanding of the various pathways through which disease resistance, flowering, and other normal plant biological processes function. Furthermore, because salicylates such as aspirin and salicylic acid appear to have advantageous properties in both plants and animals with regard to treatment and/or prevention of disease, it is possible that this discovery will provide information regarding the identification and characterization of parallel mechanisms of, for example, disease resistance in both plants and animals. This could lead to the discovery of new drugs and new forms of treatment for a list of maladies ranging from headaches to high blood pressure. Because of the discovery of this binding protein, the cloning of its gene and the growing understanding of its role in signal transduction, it may be possible to introduce into plants disease resistance mechanisms which might otherwise not be found in that species. It may also be possible to provide enhanced disease resistance, as well as other functions to plants which do use salicylic acid as a signal. The cause of world hunger could also be advanced by such discoveries because disease resistant crops could be generated with reduced incident of crop failure.
The present inventors have discovered that the binding protein that they have isolated and purified exhibits a quantitative and qualitative correlation between binding activities and the physiological activities of salicylic acid compounds. In other words, the more biologically active a salicylate is in a plant, the more tightly it will be bound by the native salicylic acid binding protein of the present invention and vice versa. Therefore, assays or tests can be developed and used as a first step in determining whether certain salicylates, either of natural or synthetic origin, might be biologically active and, therefore, agriculturally or pharmaceutically important,
The inventors have discovered that the identified salicylic acid binding protein has catalase activity which is inhibited by binding. Inhibition of catalase's H.sub.2 O.sub.2 -scavenging activity would result in an elevated level of H.sub.2 O.sub.2 and other reactive oxygen species ("ROS", also called active oxygen species--"AOS"). As used herein the term "catalase" refers to the family of enzymes known as catalases. The previously documented involvement of reactive oxygen species in host defense against microorganisms (Orlandi et al., Physiol. and Mol. Plant Pathol. 40: 173 (1992); Schwacke et al., Planta 187: 136 (1992); Apostol et al., Plant Physiol. 90: 109 (1989); Legendre et al., Plant Physiol. 102: 233 (1993); Baker et al., Plant Physiol. 102: 1341 (1993)) and the discovery that the salicylic acid binding protein is a catalase whose activity is inhibited by SA. binding suggest that the role of salicylic acid in defense may be through its modulation of the abundance of reactive oxygen species via the influencing of plant catalase activity.
This hypothesis is further supported by additional discoveries. First, the inventors have found that 2,6-dichloroisonicotinic acid (INA), which is a potent synthetic inducer of enhanced disease resistance and of proteins related to disease resistance (Uknes et al., Plant Cell 4: 645 (1992); Uknes et al., Mol Plant-Microbe Interact. 6: 692 (1993)), also inhibits catalase activity. Second, they discovered that both salicylic acid and INA also inhibit the activity of ascorbate peroxidase. As used herein "ascorbate peroxidase" refers to the family of enzymes known as ascorbate peroxidases. Since catalase and ascorbate peroxidase are the two principal enzymes in plants for destruction of H.sub.2 O.sub.2 (Bowler et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 43: 83 (1992); Jahnke et al., Plant Cell Environ. 14: 98 (1991)), their inhibition by salicylic acid, INA, or functionally similar compounds would result in elevated levels of H.sub.2 O.sub.2 and other reactive oxygen species. The fact that both a natural (salicylic acid) and synthetic (INA) inducers of disease resistance inhibit both key enzymes involved in the removal of H.sub.2 O.sub.2 illustrates that modulation of the level of reactive oxygen species is an important step in the development of disease resistance.